One of the greatest challenges in cryobiology is the cryopreservation of entire organs. Although difficult, this goal is important [14, 15, 24, 25, 28, 29], in part because present limits on human organ storage times after procurement for transplantation substantially reduce the effectiveness and increase the cost of organ replacement [15]. These problems could be eliminated if organs could be banked [9, 24, 25] and stored for times that are shorter than current organ recipient waiting times. Although organ cryopreservation has usually been conceptualized as a way of facilitating the replacement of vital organs by allografts or xenografts, there is also considerable current interest in using the technique to preserve gonads during chemotherapy and then return them to the donor after the completion of treatment [29]. Indefinite-term cryopreservation is probably also essential for solving the largest problem in transplantation medicine, which is the shortfall in organ availability in relation to the total number of transplants that are needed. To address this need, a multi-billion dollar investment in the field of tissue engineering has been made [23], but this approach will also require cryopreservation in order to achieve inventory control and efficient supply chain management of the tissue-engineered products [13].
The cryopreservation of organs was first seriously investigated in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Until 1981, it was assumed that freezing was the only option for cryopreservation, but in 1981, Fahy introduced the radically different concept of vitrification, in which no ice is allowed to form in the organ during either cooling or warming, thus eliminating mechanical injury from ice. In 1985, Rall and Fahy [27] coined the term “vitrification solution,” which is a cryoprotectant solution concentrated enough to permit vitrification on cooling and, preferably, no devitrification (freezing) on rewarming after previous vitrification. Although it is thought that any aqueous sample that can be cooled at ultrarapid rates can be vitrified in principle, in the context of organ vitrification, or even in the context of the vitrification of small biological systems like embryos that are to be cooled and warmed in containers, a vitrification solution must be concentrated enough to vitrify when cooled at, generally, less than 3,000° C./min.
Human kidneys, for example, can be cooled no more rapidly than 2° C./min in their core, and for such a case a vitrifiable concentration of cryoprotectant would be defined as a concentration that allows vitrification in a kidney-sized object cooled at 2° C./min or less. Generally, “vitrification” in this context means that no, or at most very few, visible ice crystals would form in such a volume on cooling. Means of cooling organs more rapidly by vascular perfusion with cold heat exchange media would relax the definition of “vitrifiable concentration” to slightly lower concentrations, but very high concentrations would still be required. Moreover, in the context of organ vitrification, it will generally be true that a vitrifiable concentration that does not permit the complete or near-complete suppression of devitrification on rewarming at practicable warming rates will not be useful because devitrification on warming may be unacceptably damaging. As used herein, a “vitrifiable concentration” is defined as a concentration that is capable of allowing vitrification at a cooling rate of ≦20° C./min as judged by visual absence of ice in a 10 ml sample after cooling to below the glass transition temperature (Tg) or by absence of detectable exotherms when the solution is cooled to below Tg in a differential scanning calorimeter (DSC).
Since the introduction of the concept of organ vitrification, many advances have been made in the art. However, as of 2004, 23 years have passed since the concept of organ vitrification was first suggested [3], and 19 years have passed since the first proof-of-principle experiment was published showing that mammalian embryos can be vitrified and rewarmed with high survival [27], yet the original goal of successfully vitrifying organs remains elusive.
Processes related to the cryopreservation of organs, including methods and compositions for the introduction and removal of vitrifiable concentrations of cryoprotective agents, have been described in the prior art. For example, U.S. Pat. Nos. 5,723,282 and 5,962,214 claim the following method for preparing organs, tissues, or cells for vitrification:                a) cryoprotectant concentration is gradually elevated to a first concentration while the temperature is mildly reduced;        b) the first concentration is maintained for a sufficient time to permit the approximate osmotic equilibration of the organ or tissue (defined as <50-200 mM difference between arterial and venous concentrations for organs) to occur;        c) concentration is raised to a first intermediate concentration that is not sufficient to permit vitrification (is not vitrifiable);        d) the first intermediate concentration is maintained for a sufficient time to permit the approximate osmotic equilibration of the organ or tissue with the non-vitrifiable intermediate concentration (<50-200 mM difference between arterial and venous concentrations for organs);        e) the temperature is further reduced; and        f) the concentration of cryoprotectant is increased to a level sufficient for vitrification, or to a level still insufficient for vitrification followed by an additional cooling step and a final step of increasing concentration to a final, vitrifiable concentration.        
U.S. Pat. Nos. 5,821,045 and 6,187,529 claim a method in which a previously cryopreserved organ is:                a) warmed without perfusion to a temperature high enough to permit reperfusion of the organ wherein damage is minimized, and then        b) perfused directly with a composition comprising a non-vitrifiable concentration of cryoprotectant that is less than the concentration of cryoprotectant used for cryopreservation, and further comprising one or two osmotic buffering agents, where an osmotic buffering agent is defined as an extracellular solute that counteracts the osmotic effects of greater intracellular and extracellular concentrations of cryoprotectants during the cryoprotectant efflux process. When a liver is being treated, osmotic buffering agents are omitted, but step b) still requires perfusing the liver with a non-vitrifiable concentration of cryoprotectant immediately after attaining the target reperfusion temperature. According to the process limits of the prior art, the concentration during step b) is limited to 20-40% w/v or to about 3-6M, or 60% of the highest concentration perfused.        
Clearly, the prior art of adding and removing cryoprotectants and for cooling and warming has proven inadequate for organs as evidenced by the lack of any actual demonstrated success after cooling organs to cryogenic temperatures and rewarming them. Thus, while U.S. Pat. No. 6,395,467 B1 and U.S. patent application Ser. No. 09/916,396 provide extraordinary vitrification solutions and an excellent carrier solution for enhancing their effectiveness, there is still a need in the art for further improvements in the methods and compositions employed for adding and removing cryoprotectants and for cooling and warming organs and tissues.